The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module, and also establishes a still functionally not characterized interaction with {b}-Amyloid Precursor Protein (APP). Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 become hyperphosphorylated following activation of stress and MAP kinases. By immobilized metal affinity chromatography and a combined {m}LC/MALDI-TOF/ESI-IT mass spectrometry approach, we have identified thirty-five sites of mitotic phosphorylation within JIP1, among which eight present within Ser/Thr-Pro sequence. This motif is modified by various kinases also in aggregates of the microtubule-associated protein tau, which generate typical intra-neuronal lesions occurring in Alzheimer's disease. Most of the post-translational modifications found were located within the JNK, MAP-KK and RAC- Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phospho-tyrosine interaction domains, which are essential for binding to kinesin, APP and MAP-KKK. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress and MAP kinases indicate that these signalling pathways might modulate JIP1 signalling by regulating its stability and association with some, but not all, interacting proteins.